Philip Roelandt

Human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis C virus

BACKGROUND & AIMS Worldwide, about 180 million people are chronically infected with the hepatitis C virus (HCV). Current in vitro culture systems for HCV depend chiefly on human hepatoma cell lines. Although primary human hepatocytes support HCV infection in vitro, and immunodeficient mice repopulated with human hepatocytes support HCV infection in vivo, these models are limited because of shortage of human livers to isolate hepatocytes. Therefore, there is significant interest in the establishment from of a HCV culture system in human stem cell-derived hepatocyte-like cells. METHODS Human embryonic stem cell (hESC)-derived hepatocytes were infected with HCV in the presence or absence of direct acting antivirals. After inoculation, replication of HCV was analyzed extensively. RESULTS We demonstrate that hESC-derived hepatocytes can be infected with the HCV JFH1 genotype 2a, resulting in the production of viral RNA in the stem cell progeny. Viral replication is inhibited by a non-nucleoside HCV polymerase-inhibitor (HCV-796), a cyclophilin binding molecule (Debio 025-Alisporivir) and the protease inhibitor VX-950 (Telaprevir). Stem cell-derived hepatocytes produced, for more than 10 days, the HCV core protein as well as virions that were capable of re-infecting hepatoma cells. CONCLUSIONS Hepatocytes derived from hESC support the complete HCV replication cycle (including the production of infectious virus), and viral replication in these cells is efficiently inhibited by selective inhibitors of HCV replication.

Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm, and Finally Hepatocyte-Like Cells

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development.

Directed differentiation of murine-induced pluripotent stem cells to functional hepatocyte-like cells

BACKGROUND & AIMS Induced pluripotent stem (iPS) cells exert phenotypic and functional characteristics of embryonic stem cells even though the gene expression pattern is not completely identical. Therefore, it is important to develop procedures which are specifically oriented to induce iPS cell differentiation. METHODS In this study, we describe the differentiation of mouse iPS cells to hepatocyte-like cells, following a directed differentiation procedure that mimics embryonic and fetal liver development. The sequential differentiation was monitored by real-time PCR, immunostaining, and functional assays. RESULTS By sequential stimulation with cytokines known to play a role in liver development, iPS cells were specified to primitive streak/mesendoderm/definitive endoderm. They were then differentiated into two types of cells: those with hepatoblast features and those with hepatocyte characteristics. Differentiated hepatocyte-like cells showed functional properties of hepatocytes, such as albumin secretion, glycogen storage, urea production, and inducible cytochrome activity. Aside from hepatocyte-like cells, mesodermal cells displaying some characteristics of liver sinusoidal endothelium and stellate cells were also detected. CONCLUSIONS These data demonstrate that a protocol, modeled on embryonic liver development, can induce hepatic differentiation of mouse iPS cells, generating a population of cells with mature hepatic phenotype.

Differentiation of rat multipotent adult progenitor cells to functional hepatocyte-like cells by mimicking embryonic liver development

Differentiation of stem cells to hepatocytes has industrial applications, as well as the potential to develop new therapeutic strategies for liver disease. The protocol described here, sequentially using cytokines that are known to have a role in liver embryonic development, efficiently differentiates rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells by directing them through defined embryonic intermediates, namely, primitive streak/mesendoderm/definitive endoderm, hepatoblast and hepatocyte-like phenotype. After 20 days, the final differentiated multipotent adult progenitor cell progeny is a mixture of cells, comprising cells with the characteristics of hepatoblasts and a smaller cell fraction with the morphological and phenotypical features of mature hepatocytes, as well as other mesodermal cells and some persistent undifferentiated rMAPCs. A detailed functional characterization of the stem cell progeny is also described; this should be used to confirm that differentiated cells display the functional characteristics of mature hepatocytes, including albumin secretion, glycogen storage and several detoxifying functions such as urea production, bilirubin conjugation, glutathione S-transferase activity and cytochrome activity.

Hepatosplenic γδ T-cell lymphoma after liver transplantation: Report of the first 2 cases and review of the literature

Hepatosplenic γδ T‐cell lymphoma is a rare lymphoproliferative disorder originating from natural killer–like Vδ1‐lymphocytes. This subtype has been described after different types of solid organ transplants. In this article, we describe the first 2 cases after liver transplantation. Both patients had thrombocytopenia with (hepato)splenomegaly but without peripheral lymphadenopathies and sinusoidal infiltration of the liver and spleen by monomorphic γδ‐lymphocytes on pathological examination. The clinical and pathological findings, immunophenotypical profile, prognosis, and treatment are highlighted. In order to make an early diagnosis, physicians who take care of liver transplant recipients should be aware of the characteristic features of this posttransplant lymphoproliferative disorder. Therefore, a diagnostic algorithm is proposed. Liver Transpl 15:686–692, 2009.

HNF1B deficiency causes ciliary defects in human cholangiocytes

Heterozygous deletion or mutation in hepatocyte nuclear factor 1 homeobox B/transcription factor 2 (HNF1B/TCF2) causes renal cyst and diabetes syndrome (OMIM #137920). Mice with homozygous liver‐specific deletion of Hnf1β revealed that a complete lack of this factor leads to ductopenia and bile duct dysplasia, in addition to mild hepatocyte defects. However, little is known about the hepatic consequences of deficient HNF1B function in humans. Three patients with heterozygous HNF1B deficiency were found to have normal bile duct formation on radiology and routine liver pathology. Electron microscopy revealed a paucity or absence of normal primary cilia. Therefore, heterozygous HNF1B deficiency is associated with ciliary anomalies in cholangiocytes, and this may cause cholestasis. (HEPATOLOGY 2012;56:1178–1181)

MAPC culture conditions support the derivation of cells with nascent hypoblast features from bone marrow and blastocysts

Dear Editor, We previously demonstrated (Jiang et al., 2002) that rodent multipotent adult progenitor cells (MAPC) can self-renew longterm while maintaining multilineage differentiation capacity. Rodent MAPC express a number of pluripotency-related transcription factors (TF) including Oct4 and Rex1 but not Nanog and Sox2, two other TF known to play a significant role in the maintenance of the pluripotency of embryonic stem cells (ESC) (Ulloa-Montoya et al., 2007). However, rodent MAPC express several TF, including Gata4, Gata6, Sox7 and Sox17, typically expressed in the nascent hypoblast of the developing inner cell mass (ICM) (Nichols and Smith, 2011) and in the recently described rat extrambryonic endodermal precursor cells (rXEN-P), which are isolated from blastocyst (Debeb et al., 2009). We derived in 4/12 independent isolations one or more rMAPC lines, by culturing rat BM cells in rMAPC medium (rMAPC isolation scheme, Supplementary Figure S1). After 4 weeks of culture, BM cells were depleted of CD45+ cells and 2–8 weeks later, clusters of refractile and small cells appeared, which became the preponderant cell type within 10 days (Figure 1A). Nearly all cells from the established lines expressed Oct4, Gata4, Gata6, Sox7 and Sox17 transcripts and proteins (Figure 1B and Supplementary Figure S2A and B), as well the surface markers SSEA1 and CD31 (Figure 1C and Supplementary Figure S2C), both markers of the early ICM. Although rMAPC lines express Oct4, previous studies (Lengner et al., 2007) demonstrated that Oct4+ cells cannot be detected in adult mouse tissues and that Oct4 is not required for postnatal tissue homeostasis. Based on Lengner’s findings, we hypothesized that the rMAPC phenotype could be the result of a culture-induced reprogramming. We therefore analysed BM-cultures during 2 independent rMAPC isolations before, during and after the appearance of the refractile and small cells. We could not identify any Oct4+ or SSEA1+/CD31+ cells in more than one million cells analyzed after CD45+ cells depletion (Figure 1C and D, and Supplementary Figure S2D and E), several weeks before the appearance of the refractile cells positive for these markers. RT-qPCR analysis further demonstrated that acquisition of the typical rMAPC morphology was associated with .1000fold increase in expression of Oct4 and the typical hypoblast gene transcripts (Figure 1E and Supplementary Figure S2F). Although some rMAPC lines had karyotypical abnormalities (Supplementary Figure S2G and Table S1), some lines did not, suggesting that the rMAPC phenotype is not induced by a specific translocation, duplication and/or deletion. These studies demonstrate that rMAPC do not exist in BM and that this hypoblast phenotype is acquired upon prolonged in vitro culture. rMAPC may represent a rare event of in vitro reprogramming, resembling what has been observed during spermatogonial stem cell (Guan et al., 2006; Kanatsu-Shinohara et al., 2008; Ko et al., 2009) and epiblast stem cell (Bao et al., 2009) de-differentiation to ESC-like cells, when cultured under ESC conditions. Because the gene expression pattern of rMAPC (Ulloa-Montoya et al., 2007) and rXEN-P cells (Debeb et al., 2009) is highly similar, we next asked whether BM cells were reprogrammed to a hypoblast/extraembryonic progenitor fate. To investigate this, we tested whether rMAPC could be cultured under rXEN-P conditions and vice versa. When established rXEN-P lines were cultured under rMAPC conditions for 1–2 passages, they grew dispersed, acquiring the typical rMAPC morphology (Supplementary Figure S3A). rXEN-P cells became homogeneously Oct4+/Gata4+ and the percentage of SSEA1+ cells increased (Supplementary Figure S3B and E). RT-qPCR revealed that no differences in RNA expression for hypoblast genes could be detected in XEN-P lines, once cultured in MAPC conditions, except for higher levels of Sox17 and lower levels of Tmprss2 (Supplementary Figure S3D). By contrast, when rMAPC were cultured in XEN-P medium on rat embryonic feeders, typical XEN-P colonies were generated, i.e. Oct42/Gata4+ epithelioid cells with a rim of loosely attached small refractile cells that are Oct4+/Gata4+ (Supplementary Figure S3F and G). Moreover, the percentage of SSEA1+ cells decreased (Supplementary Figure S3J); consistently, RT-qPCR revealed a decrease in Sox17 and an increase in Tmprss2 (Supplementary Figure S3I). Cell doubling time of rMAPC or rXEN-P cells cultured in MAPC conditions was slightly faster than in XEN-P conditions (Supplementary Figure S3A and F). Therefore, rMAPC culture conditions supported the feederfree growth of established XEN-P clones in a more homogenous and immature state. To further define the relationship between rMAPC, XEN-P and typical XEN cells, rMAPC and rXEN-P cells were also cultured under standard XEN conditions (Kunath et al., 2005) without exogenous LIF (Supplementary Figure S4A). rXEN-P and rMAPC cells in XEN conditions, formed extraembryonic endodermal colonies with significantly lower proliferation rate (Supplementary Figure S4B and E). Expression of hypoblast gene transcripts doi:10.1093/jmcb/mjs046 Journal of Molecular Cell Biology (2012), 4, 423–426 | 423 Published online August 9, 2012

Acute Liver Failure Secondary to Khat (Catha edulis)–Induced Necrotic Hepatitis Requiring Liver Transplantation: Case Report

We describe the case of a 26-year-old man with acute liver failure secondary to ingestion of khat (Catha edulis) leaves. In fact, this is the first case of acute liver failure due to khat reported outside the United Kingdom. The combination of specific epidemiologic data (young man of East African origin) and clinical features (central nervous system stimulation, withdrawal reactions, toxic autoimmune-like hepatitis) led to the diagnosis. Mechanisms of action and potential side effects of khat are elaborated on.

Directed Differentiation of Pluripotent Stem Cells to Functional Hepatocytes

Differentiation of human stem cells to hepatocytes is crucial for industrial applications as well as to develop new therapeutic strategies for liver disease. The protocol described here, using sequentially growth factors known to play a role in liver embryonic development, efficiently differentiates human embryonic stem cells (hESC) as well as human-induced pluripotent stem cells (hiPSC) to hepatocytes by directing them through defined embryonic intermediates, namely, mesendoderm/definitive endoderm and hepatoblast and hepatocyte phenotype. After 28 days, the final differentiated progeny is a mixture of cells, comprising cells with characteristics of hepatoblasts and a smaller cell fraction with morphological and phenotypical features of mature hepatocytes. An extensive functional characterization of the stem cell progeny should be used to confirm that differentiated cells display functional characteristics of mature hepatocytes including albumin secretion, glycogen storage, and several detoxifying functions such as urea production, bilirubin conjugation, glutathione S-transferase activity, cytochrome activity and drug transporter activity.

Heterozygous α1-antitrypsin Z allele mutation in presumed healthy donor livers used for transplantation.

Objectives The Z allele (Glu342Lys) in &agr;1-antitrypsin (AAT) deficiency is a combined deficiency and dysfunctional allele. Carrying one Z allele induces a risk of a more aggressive evolution in patients with a chronic liver disease. As most of the carriers of Z allele do not have overt liver disease, it is likely that Z allele-containing livers have been used previously for liver transplantation. We analyzed the incidence, epidemiology, and clinical features of AAT accumulation in the hepatocytes after liver transplantation. Methods Follow-up biopsies of liver transplant recipients were analyzed with periodic acid Schiff staining until 2006 (n=486); from 2006 on (n=303), all biopsies were stained with a specific monoclonal antibody against mutated AATZ protein. Genotyping of both recipient and donor was performed in the case of positive staining. Results Of 789 liver transplantation patients, six patients (0.8%) showed mutated AATZ accumulation in the transplanted liver. Mutation analysis confirmed the presence of the Z allele in all donor organs including one transplanted organ with the SZ phenotype. There was a clear concordance between the isoelectrical focusing of the recipient AAT after transplantation and the genotype of the donor. Conclusion Presumed healthy donor organs containing the Z allele were used for transplantation in 0.8% of cases in our series. As the presence of a Z allele is an independent risk factor of aggravation of chronic liver disease, AATZ accumulation in biopsies after liver transplantation should be actively looked for.